Recently, enzymes catalyzing transfer or release of modified groups involved in phosphorylation, acetylation, and lipid or sugar chain modification, and functional proteins that serve as substrate of these enzymes are regarded as targets in the development of new drugs, such as anti-cancer agents, antimicrobial agents, and antibiotics. Among these enzymes, histone deacetylase is an excellent candidate as the target for developing new anti-cancer agents. Previous reports have demonstrated that agents, such as sodium butyrate, Trichostatin A, and Trapoxin, function as histone deacetylase inhibitors. These inhibitors were originally found as anti-fungal antibiotics or substances converting the morphology of v-sis-transformed cells to normal (Taunton, J. et al., Science Vol. 272, 408-411, 1996; Yoshida, M. et al., J. Biol. Chem. Vol. 265, 17174-17179, 1990). Subsequent studies revealed that the target of these agents was the histone deacetylase. Further, histone deacetylase inhibitors having strong antineoplastic activity as follows are known in the art:    FR901228 (Fujisawa Pharmaceutical Co.)    MS275 (Mitsui Pharmaceuticals Inc.).
These inhibitors of the histone deacetylase are known to induce expression of p21CIP, which is a Cdk inhibitor protein. However, other detailed mechanism of these inhibitors to exert carcinostatic action remains to be clarified.
Histone deacetylase plays an important role in the regulation of the expression of various genes by changing the structure of nucleosomes (Davie, J. R. and Chadee, D. N., J. Cell Biochem. (Suppl.) 30-31, 203-213, 1998). The histone deacetylase also has been reported to participate in the process of cell cycle and cell differentiation, and disturbance in their regulation is involved in some types of cancers (Kouzarides, T., Curr. Opin. Genet. Dev. Vol. 9, 40-84, 1999; Fenrick, R. and Hiebert, S. W., J. Cell Biochem. (Suppl.) 30-31, 194-202, 1998). Furthermore, histone deacetylase inhibitors, such as Trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA), have been known to possess antineoplastic effect. Moreover, histone deacetylase inhibitors have been reported to possess activities as follows:    Inhibition of cell proliferation (Yoshida, M. et al., Bioassays Vol. 17, 423-430, 1995; Richon, V. M. et al., Proc. Natl. Acad. Sci. USA Vol. 93, 5705-5708, 1996; Richon, V. M. et al., Proc. Natl. Acad. Sci. USA. Vol. 95 3003-3007, 1998);    Induction of final differentiation (Yoshida, M., et al., Bioassays Vol. 17, 423-430, 1995; Richon, V. M. et al., Proc. Natl. Acad. Sci. USA. Vol. 93, 5705-5708, 1996);    Suppression of tumor growth in mouse model (Cohen, L. et al., Proc. AACR Vol. 39, 108, abstr. 736, 1998; Desai, D. et al., Proc. AACR Vol. 40, 2396, abstr. 362, 1999); and    Effect in the treatment of acute promyelocytic leukemia (Fenrick, R. and Hiebert, S. W., J. Cell Biochem. (Suppl.) 30-31, 194-202, 1998).
As described above, histone deacetylase inhibitors are expected as novel anti-cancer agents. Furthermore, they are also expected to function as antimicrobial substances. Thus, screening of histone deacetylase inhibitors are likely to be further carried out to discover substances with similar activities.
However, previous methods for measuring the activity of histone deacetylases are very complicated. More specifically, according to a known method, first, radiolabeled acetic acid is added to culture cell medium to metabolically radiolabel histone in cells. The histone purified from these cells is reacted with deacetylase. Following the reaction, radiolabeled acetyl groups released from the histone are extracted with ethyl acetate to measure the activity of the enzyme based on the radioactivity (Laherty, C. D. et al., Cell Vol. 89, 349-356, 1997; Hassig, C. et al., Cell Vol. 89, 341-347, 1997).
Method for measuring the activity of deacetylase without the use of radioactive substance has been also reported. According to the method, acetylated lysine residues, which have been labeled with a fluorescent substance, are used as substrates, and thus the measurement requires separtaion of the reaction product by reverse phase HPLC (Hoffmann, K. et al., Nucleic Acids Res. Vol. 27, 2057-2058, 1999).
On the other hand, important roles of enzymes that acetylate histone have been also discovered. Histone acetylase, also called histone acetyltransferase (HAT), has been revealed to regulate gene transcription through the acetylation of histones. Specifically, histones generally tightly coil DNAs to fold the DNAs to a compact structure. When the N-terminal basic amino acid, lysine residue, of a histone is acetylated, the binding with the DNA loosens, which is suggested to lead to mRNA synthesis. Method for measuring the activity of acetylase is basically carried out according to the same principle as those for measuring the activity of deacetylase.
As described above, the known methods comprise complicated procedures, and therefore it is difficult to treat multiple samples and to carry them out under various conditions. Thus, to readily conduct large-scale screening, such as those for discovering new drugs, a much more simple system without the use of radioisotopes have been desired.
Recently, the development and introduction of the combinatorial chemical library system, which enabled continuous fully-automatically synthesis of organic compounds as seeds of pharmaceutical agents, has realized the development of a system by which the screening of compounds can be achieved at faster speed. This system is entirely different from previous chemical libraries that comprise organic compounds extracted from natural material, and theoretically enables production of novel organic compounds without limitation. Today, major pharmaceutical companies around the world proactively introduce this innovative system. As a result, vast numbers of candidate compounds have been produced. Thus, much simpler methods for measuring activity of the compounds are intensely demanded to achieve a more efficient screening as ever for useful substances that may be used as pharmaceutical agents. However, most conventional methods are very complicated, and required high costs.
Conventional screening method includes the binding assay system. According to the system, the influence of a compound on the binding between proteins, such as enzymes, and the substrate thereof, low-molecular compounds and binding proteins, are determined. The binding assay system indeed provided a solution for the recent problem of efficiency in such screening. However, the system basically requires the use of radiolabeled substrates and huge investment for the introduction of the system. Moreover, it is basically impossible to estimate the level of the inhibitory activity of an enzyme reaction based on the system that utilizes intermolecular association as an index. In addition, radioisotopes are often used in this system, and as a consequence produces radioactive waste. Finally, a method of readily screening for compounds that regulate deacetylase activity is desired.